Volume 16, Issue 2, Pages 203-216 (February 2009) Sanguinarine Interacts with Chromatin, Modulates Epigenetic Modifications, and Transcription in the Context of Chromatin  Ruthrotha Selvi B, Suman Kalyan Pradhan, Jayasha Shandilya, Chandrima Das, Badi Sri Sailaja, Naga Shankar G, Shrikanth S. Gadad, Ashok Reddy, Dipak Dasgupta, Tapas K. Kundu  Chemistry & Biology  Volume 16, Issue 2, Pages 203-216 (February 2009) DOI: 10.1016/j.chembiol.2008.12.006 Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 1 SGR Interacts with Histones (A) Structure of SGR. (B) Curve-fitting analyses to evaluate the dissociation constant for the association of SGR with different levels of chromatin structure: chromatin (○), mononucleosome (Δ), and chromosomal DNA (□) in 10 mM Tris-HCl (pH 6.5) at 25°C. Dissociation constant values obtained from the fitting of the binding isotherms by the nonlinear least-squares method are 10 μM, 12 μM, and 17 μM for chromosomal DNA, chromatin, and mononucleosome, respectively. The concentration of SGR taken was 5 μM in all three cases. (C) Exchange of heat of association of SGR with chromatin. Upper panel is the isothermal calorimetric titration of 1.2 mM chromatin into 10 μM SGR at 25°C in 10 mM Tris-HCl buffer (pH 6.5). Lower panel is the exothermic heat exchanged per mole of injectant as a function of molar ratio of chromatin to SGR. The data were fitted with the “one set of sites” binding model. Solid line represents the fit of the binding isotherm. (D) Temperature dependence of calorimetric enthalpy of association between SGR and chromatin (○), mononucleosome (Δ), and chromosomal DNA (□). The lines represent the fits of data to equation ΔH (T) = ΔH (T0) + ΔCp∗(T-T0). Values of ΔCp obtained from the fits are −0.2, −0.09, and + 0.40 kcal mol−1 K−1 for chromatin, chromosomal DNA, and mononucleosome, respectively. (E) Temperature dependence of calorimetric entropy of association of SGR with different levels of chromatin structures: chromatin (○), mononucleosome (Δ), and chromosomal DNA (□). Chemistry & Biology 2009 16, 203-216DOI: (10.1016/j.chembiol.2008.12.006) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 2 SGR Induces Conformational Changes upon Interacting with Chromatin (A) Circular dichroism spectra of 10 μM SGR alone (…) and in presence of 39 μM polymer (---) in 10 mM Tris-HCl (pH 6.5) at 25°C. Panels I, II, and III correspond to chromatin, mononucleosome, and chromosomal DNA, respectively. Spectrum 2 (- - -) is the only polymer (39 μM) in all three cases. Inset of (A) is the plot of molar ellipticity value at 272 nm (θ272) versus time for chromatin. (B) Panel I: Fluorescence quenching of SGR in presence of histone octamer for SGR alone (5 μM, spectrum 1) and in the presence of octamer (5 μM, spectrum 2; 8 μM, spectrum 3; 16 μM, spectrum 4). Panel II: Best fit curve of the binding isotherm generated by nonlinear least-squares method for the association of SGR with octamer under previously mentioned conditions. (C) Intensity distribution (%) of different levels of chromatin structure as a function of size (in diameter) obtained from DLS measurements. Figures in the left panel represent chromatin, mononucleosome, and chromosomal DNA (from top to bottom), respectively. Figures in the right panel represent corresponding polymer complexed with SGR. All experiments were done in 10 mM Tris-HCl (pH 6.5) at 25°C. (D and E) Global chromatin organization upon treatment with SGR was probed by confocal (D) and atomic force microscopy (E). Panel DI and DII (i, ii, iii) and panel EI (i, ii, iii) are images of intact nuclei. Panel EII (iv, v, vi) are AFM images upon MNase digestion. (i), (ii), and (iii) in (D, E) represent the untreated, DMSO-treated, and 5 μM SGR-treated cells. (iv), (v), and (vi) in (E) are MNase-digested images after similar treatments. Chemistry & Biology 2009 16, 203-216DOI: (10.1016/j.chembiol.2008.12.006) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 3 SGR Inhibits Histone Methylation (A–C) HMTase assays were performed in the presence or absence of SGR using highly purified HeLa core histones (800 ng) and processed for filter binding (A) and gel assay fluorography (B, C). Lane 1, without any HMTase; lane 2, with HMTase; lane 3, with HMTase and in the presence of DMSO as solvent control; lanes 4–7, with HMTase and in the presence of 5, 10, 15, and 20 μM concentrations of SGR, respectively. (D) Histones extracted from the compound-treated cells and subjected to western blotting analysis with antibodies against methylated H3K9 (I), H3R17 (II), and H3K4 (III) antibodies. Lane 1, untreated cells; lane 2, DMSO (solvent control) treated cells; lanes 3 and 4, 1 and 2 μM SGR-treated cells. Loading and transfer of equal amounts were confirmed by immunodetection of histone H3 (IV). Chemistry & Biology 2009 16, 203-216DOI: (10.1016/j.chembiol.2008.12.006) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 4 SGR Is a Potent Inhibitor of HATs (A–C) Histone acetyltransferase assays were performed in the presence or absence of SGR using highly purified HeLa core histones (800 ng) and processed for filter binding (A) and gel assay fluorography (B, C). Lane 1, without any HAT; lane 2, with HAT; lane 3, with HAT and in the presence of DMSO as solvent control; lane 4, with HAT in the presence of garcinol (100 μM); lanes 5–7, with HAT and in the presence of 5, 10, 15, and 20 μM SGR respectively. (D) Histones extracted from the compound treated cells and subjected to western blotting analysis with antibodies against acetylated histone H3. Lane 1, untreated cells; lane 2, DMSO (solvent control) treated cells; lanes 3 and 4, 1 and 2 μM SGR-treated cells. Loading and transfer of equal amounts were confirmed by immunodetection of histone H3. (E) Upon treatment, mice liver tissue was processed for IHC analysis. Haematoxylin and eosin staining (I) and immunohistologic staining using acetylated histone H3 antibody (II) were performed. Chemistry & Biology 2009 16, 203-216DOI: (10.1016/j.chembiol.2008.12.006) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 5 SGR Does Not Affect Transcription from DNA Template but Inhibits p300 Histone Acetyltransferase-Dependent Chromatin Transcription (A–E) A schematic representation of in vitro transcription protocol. In vitro transcription from naked DNA template (B and D) and chromatin template (C and E). Freshly assembled chromatin template or equivalent amount of DNA (28 ng) was subjected to the protocol described in (A) with or without SGR. Lane 1, without activator (basal transcription); lane 2, with activator (Gal4-VP16); lane 3, with activator and DMSO; lanes 4–6, with activator and different concentration of SGR (as indicated). Chemistry & Biology 2009 16, 203-216DOI: (10.1016/j.chembiol.2008.12.006) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 6 SGR Modulates Global Gene Expression (A) Microarray analysis of gene expression upon treatment of HeLa cells with SGR. Lane 1 represents the forward reaction and lane 2 represents the dye swap. (B) Validation of differentially altered genes by using real-time polymerase chain reaction (RT-PCR). CHFR3 and PRKCH represent upregulated genes. MEF2C and DPPA2 represent downregulated genes. (C) Microarray analysis of gene expression upon treatment of HeLa cells with SGR. Lanes 1, 2, and 3 represent forward reaction, and lanes 4 and 5 represent dye swap. (D) Validation of differentially altered genes by using RT-PCR. PLIN and DIABLO represent upregulated genes. ATF6 and HMGA2 represent downregulated genes. Chemistry & Biology 2009 16, 203-216DOI: (10.1016/j.chembiol.2008.12.006) Copyright © 2009 Elsevier Ltd Terms and Conditions